Antioxidant, Antimicrobial, and Bioactive Potential of Two New Haloarchaeal Strains Isolated from Odiel Salterns (Southwest Spain)
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biology Article Antioxidant, Antimicrobial, and Bioactive Potential of Two New Haloarchaeal Strains Isolated from Odiel Salterns (Southwest Spain) Patricia Gómez-Villegas 1 , Javier Vigara 1 , Marta Vila 1, João Varela 2 , Luísa Barreira 2 and Rosa Léon 1,* 1 Laboratory of Biochemistry, Department of Chemistry, University of Huelva, Avda. de las Fuerzas Armadas s/n, 21071 Huelva, Spain; [email protected] (P.G.-V.); [email protected] (J.V.); [email protected] (M.V.) 2 Centre of Marine Sciences, University of Algarve, Campus of Gambelas, 8005-139 Faro, Portugal; [email protected] (J.V.); [email protected] (L.B.) * Correspondence: [email protected]; Tel.: +34-95-921-9951 Received: 15 August 2020; Accepted: 17 September 2020; Published: 18 September 2020 Simple Summary: Halophilic archaea are microorganisms that inhabit in extreme environments for life, under salt saturation, high temperature and elevated UV radiation. The interest in these microorganisms lies on the properties of their molecules, that present high salt and temperature tolerance, as well as, antioxidant power, being an excellent source of compounds for several biotechnological applications. However, the bioactive properties from haloarcahaea remain scarcely studied compared to other groups as plants or algae, usually reported as good health promoters. In this work we describe the isolation and the molecular identification of two new haloarchaeal strains from Odiel salterns (SW Spain), and the antioxidant, antimicrobial and bioactive potential of their extracts. The results revealed that the extracts obtained with acetone presented the highest activities in the antioxidant, antimicrobial and anti-inflammatory assays, becoming a promising source of metabolites with applied interest in pharmacy, cosmetics and food industry. Abstract: The need to survive in extreme environments has furnished haloarchaea with a series of components specially adapted to work in such conditions. The possible application of these molecules in the pharmaceutical and industrial fields has received increasing attention; however, many potential bioactivities of haloarchaea are still poorly explored. In this paper, we describe the isolation and identification of two new haloarchaeal strains from the saltern ponds located in the marshlands of the Odiel River, in the southwest of Spain, as well as the in vitro assessment of their antioxidant, antimicrobial, and bioactive properties. The acetone extract obtained from the new isolated Haloarcula strain exhibited the highest antioxidant activity, while the acetone extracts from both isolated strains demonstrated a strong antimicrobial activity, especially against other halophilic microorganisms. Moreover, these extracts showed a remarkable ability to inhibit the enzyme cyclooxygenase-2 and to activate the melanogenic enzyme tyrosinase, indicating their potential against chronic inflammation and skin pigmentation disorders. Finally, the aqueous protein-rich extracts obtained from both haloarchaea exhibited an important inhibitory effect on the activity of the acetylcholinesterase enzyme, involved in the hydrolysis of cholinergic neurotransmitters and related to several neurological diseases. Keywords: antioxidant; anti-inflammatory; antimicrobial; bioactive substances; haloarchaea Biology 2020, 9, 298; doi:10.3390/biology9090298 www.mdpi.com/journal/biology Biology 2020, 9, 298 2 of 20 1. Introduction Halophilic archaea or haloarchaea are a group of extremophilic microorganisms inhabiting hypersaline environments, such as salt lakes and salterns. They thrive in harsh conditions for life, including low water availability, high salt concentration, and elevated solar irradiance, which implies high temperature and ultra-violet (UV) radiation [1,2]. The biotechnological interest in the haloarchaeal group has increased due to their ability to produce a wide variety of compounds such as exopolysaccharides, carotenoids, and proteins adapted to work in such extreme conditions. The tolerance of these enzymes to elevated saline concentrations and temperatures, the high antioxidant power and therapeutic potential of haloarchaeal carotenoids or the good jellifying properties and thermal stability of the exopolysacharides secreted by many archaea make these metabolites highly appreciated for numerous biotechnological applications, including biomedical, pharmaceutical, cosmetic, environmental or industrial purposes [3,4]. Furthermore, considering that only a small part of the existing archaeal species has been discovered and studied, it is expected that many other metabolites with unexplored bioactivities can be obtained from this extraordinary group of microorganisms, as pointed in recent reviews [5]. Haloarchaea have shown to be particularly suitable for the production of extremozymes, which can tolerate high temperatures and saturating salt concentrations, conditions required in food, detergent and textile sectors [6,7]. In addition, a common feature within the haloarchaeal group is their ability to secrete halocins, which are peptides or proteins able to inhibit the growth of susceptible microorganisms living in the same habitat [8,9]. These molecules are also resistant to extreme conditions and could be a source of new antimicrobial compounds. Moreover, most haloarchaea can produce and accumulate carotenoids, which are responsible for their red color. Carotenoids are antioxidant compounds with an important role as human health enhancers, and it is widely known that their regular consumption helps to prevent many degenerative diseases, such as neurodegeneration, cancer, cardiovascular diseases, macular degeneration and cataracts [10,11]. Carotenoids from haloarchaea have shown anticancer and antihemolytic activities [12], and the ability to increase sperm cells viability after cryopreservation [13]. Furthermore, many natural carotenoids are used as food preservatives and pharmaceutical and cosmetic compounds due to their antioxidant and pro-vitamin properties [14]. Bacterioruberin (BR) and its derivatives, monoanhydrobacterioruberin (MABR) and bisanhydrobacterioruberin (BABR), are the main carotenoids synthesized by halophilic archaea [15–17]. These carotenoids have 50 carbon atoms (C50) and possess a longer system of conjugated double bonds than the C40 carotenoids usually found in other organisms such as plants, microalgae, fungi and bacteria [14]. Some C40 carotenoids, such as phytoene, lycopene and β-carotene have also been found in haloarchaea, but at such low quantities that they have been proposed as metabolic intermediates for the biosynthesis of C50 carotenoids [18,19]. Even though carotenoids play a unique important role in cell protection against oxidative damage, bacterioruberin appears also to be a stabilizer of the cell membrane under osmotic stress [20]. Because of the structure of bacterioruberin, it can be inferred that its antioxidant potential might be even higher than that of β-carotene, due to the fact that it has 13 conjugated double bonds and four hydroxyl groups, compared to the 9 conjugated double bonds and none hydroxyl group of β-carotene [16]. For those reasons, the antioxidant power of haloarchaeal extracts and their potential as alternative to current food preservatives and as drug leads should be more deeply studied. Pigments from different kinds of marine microorganisms have shown effective antibacterial properties. Examples of this are the red pigment prodigiosin, isolated from the sponge-associated bacterium Serratia marcescens [21]; or the chlorophyll a derivatives obtained from the microalga Isochrysis galbana [22]. Recently, the antimicrobial activity of pigments from some halophilic bacteria has been tested against bacteria and fungi [23]; however, so far, the studies about the antimicrobial activity of haloarchaeal pigments or extracts thereof are scarce [19,24]. The main aim of this work is to explore the bioactive properties of different extracts obtained from two new strains of haloarchaea isolated from the Odiel solar saltern (SW Spain), focusing on their Biology 2020, 9, 298 3 of 20 antioxidant and antimicrobial activities; and their capability to modulate the activity of disease-related enzymes involved in melanin biosynthesis, neurological degeneration, carbohydrate metabolism, and inflammatory response, through in vitro assays. 2. Materials and Methods 2.1. Sample Collection Water samples were collected from a crystallizer pond located in the natural reserve of the Odiel Marshlands, in the southwest of Spain (Latitude: 37.2395, longitude: 6.95287). The ionic composition − and the physicochemical parameters of the water brine were determined by the standard methods, as previously reported [20]. 2.2. Archaea Isolation The biomass from 2 L of the collected water samples was harvested by centrifugation at 19,800 g, × resuspended in 20 mL of archaeal medium (ATCC 1176 medium) and used for the isolation of archaeal colonies on agar plates. Typically, 1 mL of serial dilution corresponding to 1:10, 1:100 and 1:1000 were spread on agar medium containing 20% of salt. After incubation at 37 ◦C for 15 days, red and pink colonies were visible. Selected colonies were purified by at least three streaking rounds on fresh agar plates. The isolates were preserved in 20% glycerol (w/v) at 80 C for further use. − ◦ 2.3. Preliminary Selection of Archaeal Strains with Antimicrobial Activity Halocin activity was determined by observing growth inhibition of the archaea Haloferax lucetense (CECT 5871), which was purchased from CECT (Spanish Collection